Apr 13, 2026

Cell Culture Transfection and Immunoblotting

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbj88yvpk/v1
  • Roberta Marongiu1,
  • Sabina Marciano1
  • 1Weill Cornell Medical College
Jacquelyn Haytayan
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.81wgbj88yvpk/v1
Protocol Citation: Roberta Marongiu, Sabina Marciano 2026. Cell Culture Transfection and Immunoblotting. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbj88yvpk/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 13, 2026
Last Modified: April 13, 2026
Protocol  Integer ID: 314933
Keywords: ASAPCRN, cultivation of rat dopaminergic n27 cell, rat dopaminergic n27 cell, cell culture transfection, cell, transient transfection, cultivation
Funders Acknowledgements:
Aligning Science Across Parkinson's
Collaboration Research Network
Abstract
This protocol describes the cultivation of rat dopaminergic N27 cells, transient transfection and immunoblotting
Materials
Cell Culture & Transfection
  • N27 Cells: Rat dopaminergic cell line.
  • Culture Medium: DMEM (Gibco) supplemented with 10% FBS (Sigma-Aldrich) and 1% Penicillin-Streptomycin (Gibco).
  • Transfection Reagent: FuGENE6 (Promega, Cat# E2691).
  • Plasmid: pAAV DNA.

Lysis & Protein Assay
  • Lysis Buffer: * 150mM NaCl
50mM Tris pH 7.5
2mM EDTA
Protease and phosphatase inhibitor cocktail (add fresh).
  • BCA Protein Assay Kit.

Electrophoresis & Immunoblotting
  • TBS-Tween (TBST): 150 mM NaCl, 50 mM Tris (pH 7.5), and 0.1% Tween-20.
  • Blocking Buffer: 5% non-fat milk in TBST.
  • Membrane: PVDF.
  • Detection: Lumigen chemiluminescent reagents.

Equipment
  • CO2 Incubator: Set to 37degrees, 95% humidity, and 5% CO2 .
  • Imaging System: VersaDoc MP 4000 (Bio-Rad).
  • Software: Quantity One (Bio-Rad).
Cell Culture and Seeding
Maintain N27 cells in complete DMEM medium
Incubate at 37 °C in 5% CO2 and 95% humidified air
Seed cells to appropriate density prior to transfection.
Transient Transfection
Perform transient DNA transfection of pAAV using FuGENE6 reagent.
Follow the specific manufacturer’s protocol (Promega) for reagent-to-DNA ratios.
Incubate cells for 48 hours post-transfection.
Cell Lysis and Sample Preparation
Aspirate medium and wash cells with PBS.
Lyse cells using the prepared Lysis Buffer (chilled).
Centrifuge lysates to remove debris and collect the supernatant (whole cell lysate).
Quantify protein concentration using a BCA assay.
Prepare samples by normalizing to an equal amount of protein (100ug per sample).
SDS-PAGE and Transfer
Load 100ug of protein into each lane of an SDS-PAGE gel.
Run electrophoresis at desired voltage.
Transfer proteins from the gel to a PVDF membrane.
Immunoblotting
Block the membrane with 5% milk in TBST for 1 hour at room temperature.
Incubate with primary antibodies in blocking buffer or TBST overnight at 4 degrees.
Wash the membrane with TBST (typically 3 times for 10 minutes).
Incubate with HRP-coupled secondary antibodies for 1 hour at room temperature.
Wash the membrane again with TBST.
Detection and Imaging
Apply Lumigen chemiluminescent reagents to the membrane according to the manufacturer’s instructions.
Capture images using the VersaDoc MP 4000 system.
Analyze protein band density using Quantity One software.